here's one for the PCR experts...
I am trying to do a pcr reaction which requires a 72 deg. soak (3')
before the addition of TAQ. What I would do is let the machine run the
3', 72 deg. pause it, add the taq, and unpause it. The parameter is as
follows:
72 deg, 3'
72 deg, 5'
95 deg, 1'
72 deg, 3'
back to 95 deg for 20 cycles
72 deg, 10'
4 deg soak.
The problem iswhen I do one tube of this
reaction, the product looks fine. But, when I do multiple tubes (5 and
7), I get nothing. I've tried to make the technique as consistent as I
can.
Keep in mind the parameter doesn't appear to be the problem because I did
get the reaction to work, using the same parameter. And doing all the
tubes individually is not a likely option because I need about 30 tubes
of this stuff. Also, I am doing 200ul reaction in the small variety of
PCR tube...is that going to be a problem?
Thank you...
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Edward H. Wang *
Staff Resch. Assc. *
Dept. Internal Med. -Nephrology*
UC Davis *
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